Water soluble tri-substituted 1, 2-dioxetane compounds having increased storage stability, synthetic processes and intermediates

ABSTRACT

Stable, enzymatically triggered chemiluminescent 1,2-dioxetanes with improved water solubility and storage stability are provided as well as synthetic processes and intermediates used in their preparation. Dioxetanes further substituted with two or more water-solubilizing groups disposed on the dioxetane structure and an additional fluorine atom or lower alkyl group provide superior performance by eliminating the problem of reagent carryover when used in assays performed on capsule chemistry analytical systems. These dioxetanes display substantially improved stability on storage. Compositions comprising these dioxetanes, a non-polymeric cationic surfactant enhancer and optionally a fluorescer, for providing enhanced chemiluminescence are also provided.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a divisional application of application Ser.No. 09/506,263 filed Feb. 17, 2000, now U.S. Pat. No. 6,245,928 which isa continuation of Ser. No. 09/101,331, now U.S. Pat. No. 6,036,892. The'331 application resulted from National Stage Entry of PCT ApplicationUS97/19618 filed on Nov. 7, 1997. The latter PCT application is acontinuation-in-part of U.S. application Ser. No. 08/748,107, filed onNov. 8, 1996 now issued as U.S. Pat. No. 5,721,370, which is acontinuation-in-part of application Ser. No. 08/509,305 filed Jul. 31,1995 now issued as U.S. Pat. No. 5,777,135.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates generally to stable 1,2-dioxetanes andcompositions which can be triggered by chemical reagents, includingenzymes, to generate chemiluminescence. The dioxetanes contain more thanone ionizable group which are part of an alkoxy substituent. Thedioxetanes further contain a fluorine atom or lower alkyl groupsubstituted for one of the hydrogen atoms on the alkoxy substituentwhich improve the storage stability of the dioxetane. The presentinvention, in particular, further relates to methods of synthesis ofsuch dioxetanes.

The dioxetanes which are prepared by the synthetic processes of thepresent invention are useful in compositions containing the dioxetane, acationic surfactant and optionally a fluorescer which enhance the amountof chemiluminescence which is produced. Dioxetanes and enhancedcompositions of the present invention are useful in methods forgenerating light (chemiluminescence) and in methods of analysis fordetecting the presence or amount of an analyte. Importantly, theionizable groups afford a more water soluble dioxetane and solve anunexpected chemical carryover problem in capsule chemistry analyticalsystems, while the presence of the fluorine atom or lower alkyl groupimproves the storage stability of the dioxetane.

(2) Description of Related Art

a. Enzymatically Triggerable Dioxetanes.

The first examples of enzymatic triggering of dioxetanes are describedin a U.S. patent application (A. P. Schaap, U.S. patent application Ser.No. 887,139) and a series of papers (A. P. Schaap, R. S. Handley, and B.P. Giri, Tetrahedron Lett., 935 (1987); A. P. Schaap, M. D. Sandison,and R. S. Handley, Tetrahedron Lett., 1159 (1987) and A. P. Schaap,Photochem. Photobiol., 47S, 50S (1988)). The highly stableadamantyl-substituted dioxetanes bearing a protected aryloxidesubstituent are triggered to decompose with emission of light by theaction of both an enzyme and aqueous buffer to give a stronglyelectron-donating aryloxide anion which dramatically increases the rateof decomposition of the dioxetane. As a result, chemiluminescence isemitted at intensities several orders of magnitude above that resultingfrom slow thermal decomposition of the protected form of the dioxetane.U.S. Pat. No. 5,068,339 to Schaap discloses enzymatically triggerabledioxetanes with covalently linked fluorescer groups decomposition ofwhich results in enhanced chemiluminescence via energy transfer to thefluorescer. U.S. Pat. Nos. 5,112,960 and 5,220,005 and a PCT application(WO88/00695) to Bronstein disclose triggerable dioxetanes bearingsubstituted adamantyl groups. U.S. Pat. No. 4,952,707 to Edwardsdiscloses phosphate-substituted dioxetanes. A PCT application(W094/26726) to Bronstein discloses adamantyl dioxetanes bearing aphenyl or naphthyl group substituted at a non-conjugated position withan enzyme labile OX group and with an additional group on the aryl ring.

Other triggerable dioxetanes are disclosed in a PCT application(WO94/10258) to Wang. The dioxetanes disclosed in Wang contain an alkoxygroup which may be mono-substituted and a substituted phenyl-OX groupwherein one or more non-hydrogen groups are present on the benzene ringsubstituent in addition to the triggerable OX group.

Dioxetanes disclosed in all of the foregoing publications generate alight-emitting carbonyl compound comprising an alkyl ester of anaromatic carboxylic acid, typically the methyl ester of a hydroxybenzoicor hydroxynaphthoic acid or else a hydroxyaryl ketone.

Applicants' co-pending U.S. application Ser. No. 08/509,305 ('305application) filed on Jul. 31, 1995 discloses disubstituted dioxetaneswhose hydroxy dioxetane shows improved water solubility and is fullyincorporated herein by reference.

b. Surfactant Enhancement of Chemiluminescence from TriggerableDioxetanes.

Enhancement of chemiluminescence from the enzyme-triggered decompositionof a stable 1,2-dioxetane in the presence of water-soluble substancesincluding an ammonium surfactant and a fluorescer has been reported (A.P. Schaap, H. Akhavan and L. J. Romano, Clin. Chem., 35(9), 1863(1989)). Fluorescent micelles consisting of cetyltrimethylarnmoniumbromide (CTAB) and 5-(N-tetradecanoyl)amino-fluorescein capture theintermediate hydroxy-substituted dioxetane and lead to a 400-foldincrease in the chemiluminescence quantum yield by virtue of anefficient transfer of energy from the anionic form of the excited stateester to the fluorescein compound within the hydrophobic environment ofthe micelle.

U.S. Pat. Nos. 4,959,182 and 5,004,565 to Schaap describe additionalexamples of enhancement of chemiluminescence from chemical and enzymatictriggering of stable dioxetanes in the presence of micelles formed bythe quaternary ammonium surfactant CTAB. Fluorescent micelles alsoenhance light emission from the base-triggered decomposition of hydroxy-and acetoxy-substituted dioxetanes.

U.S. Pat. No. 5,145,772 to Voyta discloses enhancement of enzymaticallygenerated chemiluminescence from 1,2-dioxetanes in the presence ofpolymers with pendant quaternary ammonium groups alone or admixed withfluorescein. Other substances reported to enhance chemiluminescenceinclude globular proteins such as bovine albumin and quaternary ammoniumsurfactants. Other cationic polymer compounds were marginally effectiveas chemiluminescence enhancers; nonionic polymeric compounds weregenerally ineffective and an anionic polymer significantly decreasedlight emission. A PCT application (WO 94/21821) to Bronstein describesthe use of mixtures of the aforementioned polymeric quaternary ammoniumsurfactant enhancers with enhancement additives.

The enhancement and catalysis of a non-triggerable dioxetane by pyraninein the presence of CTAB is described (Martin Josso, Ph.D. Thesis, WayneState University (1992), Diss. Abs. Int., Vol. 53, No. 12B, p. 6305).

U.S. Pat. No. 5,393,469 to Akhavan-Tafti discloses enhancement ofenzymatically generated chemiluminescence from 1,2-dioxetanes in thepresence of polymeric quaternary phosphonium salts optionallysubstituted with fluorescent energy acceptors.

European Patent Application Serial No. 94108100.2 discloses enhancementof enzymatically generated chemiluminescence from 1,2-dioxetanes in thepresence of dicationic phosphonium salts. No documents disclose thecombination of an anionic fluorescer and a dicationic enhancer forenhancing chemiluminescence from a triggerable dioxetane. No example ofenhancement of substituted dioxetanes of the type of the presentinvention has been reported.

c. Triggerable Dioxetanes with Improved Water Solubility.

The enzymatically triggerable dioxetanes are now undergoing widespreaduse as substrates for marker enzymes in numerous applications includingimmunoassays, gene expression studies, Western blotting, Southernblotting, DNA sequencing and the identification of nucleic acid segmentsin infectious agents. Despite the growing use of these compounds, thereare limitations to there use in some assay methods. Triggerabledioxetanes whose hydroxy dioxetane deprotected form are morewater-soluble are desirable. As shown in the structures below, it isespecially desirable that the hydroxy dioxetane formed by thedephosphorylation of a phosphate dioxetane by alkaline phosphatase behighly soluble in aqueous solutions and in compositions containingchemiluminescence enhancing substances. Such dioxetanes and compositionsare of importance in certain solution assay methods for detectinghydrolytic enzymes or conjugates of hydrolytic enzymes.

As further background of the present invention and as more fullyexplained in the examples below, it has been found that use ofconventional chemiluminescent dioxetane reagents in assays performed onautomated instrumentation based on the principles of capsule chemistryanalysis results in carryover of reagent from one fluid segment toanother, resulting in potentially inaccurate measurements, erroneousresults, and imprecision due to non-reproducibility. Capsule chemistryanalysis is described in U.S. Pat. No. 5,399,497, which is fullyincorporated by reference herein. It has been postulated that, amongother possible means for overcoming the carryover problem, improvedwater solubility of the hydroxy dioxetane, in particular, mighteliminate or minimize carryover of this luminescent reactionintermediate into adjacent fluid segments of a capsule chemistryanalysis system.

Dioxetane compounds in commercial use do not incorporate anysolubilizing groups which are appended to an alkoxy group. As such,these dioxetanes are unsuitable for use in assay methods requiring zerocarryover. A suggestion of incorporating a solubilizing group into adioxetane has been made (U.S. Pat. No. 5,220,005). A dioxetane with acarboxyl group substituted on an adamantyl substituent is claimed,however, the preparation of such a dioxetane is not described.Significantly, there is no disclosure of what effect the addition of acarboxyl group had, if any, on solubility and other properties of thedioxetane. There is no teaching in the art of how many solubilizinggroups are required or what particular advantage might be conferred. Useof solubilizing groups which interfere with the removal of theprotecting group which initiates light emission or which otherwiseinterfere with light production would be of no value. Solubilizinggroups which would be removed during the luminescent reaction likewisewould not be useful.

In Applicant's co-pending '305 application it was demonstrated thatincorporation of one ionic solubilizing group was insufficient toeliminate the carryover problem associated with the hydroxy dioxetaneproduced by dephosphorylation of a phosphate dioxetane. Phosphatedioxetanes whose hydroxy dioxetane product is highly water soluble andenhanced compositions containing such phosphate dioxetanes were providedto solve this problem. It was subsequently discovered that dioxetaneswhich provided the solution to the carryover problem, exhibitedinsufficient storage stability at room temperature. Thus, no dioxetanesknown in the art possessed both high solubility of the hydroxy dioxetaneand long term storage stability.

Applicants' 08/748,107 application disclosed that substitution of ahydrogen atom on the alkoxy group bearing two ionic solubilizing groupswith a fluorine atom or lower alkyl group dramatically improves thestorage stability of these dioxetanes. Synthetic processes for preparingsuch dioxetanes were disclosed. In the present application, improvedprocesses are disclosed as well as intermediates useful therein.

OBJECTS

It is an object of the present invention to provide enzymaticallytriggered 1,2-dioxetanes with improved storage stability whose hydroxydioxetane product formed upon action of a triggering enzyme is highlysoluble in aqueous solution. It is a second object of the presentinvention to provide 1,2-dioxetanes substituted with two or morewater-solubilizing ionic groups and either a fluorine atom or loweralkyl group disposed on an alkoxy substituent of the dioxetane structurewhich provide superior storage stability. It is a further object of thepresent invention to provide a composition comprising a fluorine orlower alkyl group-substituted dioxetane with two or more ionicwater-solubilizing groups, a non-polymeric cationic enhancer andoptionally a fluorescer, for providing enhanced chemiluminescence. It isa further object of the present invention to provide dioxetanes andcompositions which, when used in assays performed on capsule chemistryanalytical systems, eliminate the problem of reagent carryover and haveextended storage stability. It is yet another object of the presentinvention to provide a synthetic process and intermediates usefultherein for the preparation of 1,2-dioxetanes substituted with two ormore water-solubilizing ionic groups and either a fluorine atom or loweralkyl group disposed on an alkoxy substituent of the dioxetanestructure.

IN THE DRAWINGS

FIG. 1 is a diagram of a capsule chemistry analysis system in whichcarryover was determined to be a problem.

FIG. 2 is a profile of adjacent segments in the capsule chemistryanalysis system showing the observed luminescence attributed tocarryover as more fully described in the Examples below.

FIG. 3 is a further profile of adjacent segments observed in theexperiments which are more fully described in the Examples below andwhich established that the carryover was not optical in nature.

FIG. 4 is a further profile of adjacent segments observed in theexperiments which are more fully described in the Examples below andwhich established that the carryover was in fact chemical in nature.

FIG. 5 is a graph depicting the relative rates of decomposition at 25°C. of a fluoro-substituted dioxetane, a chloro-substituted dioxetane, amethyl-substituted dioxetane and a reference dioxetane containing nohalogen atoms.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to dioxetanes with improved storagestability and whose hydroxy dioxetane product formed upon action of atriggering enzyme is highly soluble in aqueous solution and which aretriggerable by an enzyme to produce chemiluminescence. Such triggerabledioxetanes eliminate or minimize carryover of the luminescent hydroxydioxetane into adjacent segments in capsule chemistry analytical systemsas described in U.S. Pat. No. 5,399,497. Carryover can result fromsolubilization, deposition or precipitation of light-emitting materialof low water solubility into the fluorocarbon oil which serves as theisolating fluid in capsule chemistry systems. Reagent carryover can leadto inaccurate measurements, erroneous results and imprecision due toirreproducibility.

In the co-pending '305 application it was discovered that dioxetane 1below was particularly effective for the chemiluminescent detection ofalkaline phosphatase in aqueous solution.

For comparison, dioxetane 2 which incorporates only one ionizable groupwas prepared. This dioxetane did not eliminate the carryover problemdiscussed above.

Use of dioxetane 1 in the test system described in U.S. Pat. No.5,399,497 led to complete elimination of the carryover problem. However,it was subsequently discovered unexpectedly, that solutions of dioxetane1 in aqueous buffer displayed unsatisfactory storage stability.Solutions containing 1 in alkaline buffer displayed significantdecomposition after storage at 25° C. for two weeks. Dioxetane 1, infact, was found to be significantly less stable than a related compound,Lumigen PPD, shown below which has no ionic solubilizing groups on thealkoxy group.

As far as Applicants are aware, there is no teaching in the art ofdioxetane chemistry of the cause of the lower stability of 1. Means ofstructurally modifying 1 to improve its storage stability whilepreserving its other beneficial properties were disclosed in Applicants,co-pending application Ser. No. 08/748,107 which is fully incorporatedherein by reference.

Definitions:

Storage stability is related to the rate of decomposition of thedioxetane due to spontaneous reaction and is an intrinsic property.Decomposition of triggerable dioxetanes can also be induced by thepresence of trace quantities of agents which catalyze the removal of aprotecting group and thus initiate the decomposition. Storage stabilityof a dioxetane can be assessed by measuring the quantity of dioxetanepresent in a known sample at periodic intervals. The measurement cantake any form known which measures a property relatable to the quantityof dioxetane. Techniques such as spectrophotometry, NMR spectrometry andthe like are exemplary. A convenient means is to measure the amount oflight produced by reacting a known quantity of dioxetane with atriggering agent under a standard set of conditions. A decrease in theamount or intensity of light emitted signals a loss of dioxetanecompound.

Storage stability refers to stability of the dioxetane in both the pureform and as a solution or formulation in a buffer solution. Theformulation can also contain various additives for increasing the amountof light produced or for improving the activity of an enzymatictriggering agent. It is desirable that the dioxetane in a formulationnot undergo significant decomposition at ambient temperature for areasonable period of time. Compositions to be used with automatedanalyzers should desirably be stable for at least 1 week. Uponrefrigeration at 0-5° C., it is desirable that no significantdecomposition is observed for at least 2-3 months. More desirably,compositions to be used with automated analyzers should show not morethan 2-3% change in the observed indicator of storage stability in about2-4 weeks.

The solution to the problem of storage stability was found in dioxetaneshaving the formula I:

wherein Z is selected from the group consisting of a fluorine atom andan alkyl group of 1-4 carbons and M is selected from hydrogen, an alkalimetal ion or a quaternary ammonium or phosphonium ion, wherein R₃ and R₄are each selected from acyclic, cyclic and polycyclic organic groupswhich can optionally be substituted with heteroatoms and which providestability to the dioxetane, wherein R₂ is an aryl ring group selectedfrom phenyl and naphthyl groups which can include additionalsubstituents selected from halogens, alkyl, substituted alkyl, alkoxy,substituted alkoxy, carbonyl, carboxyl, amino and alkylamino groups andwherein X is a protecting group which can be removed by an activatingagent to form an oxyanion-substituted dioxetane which decomposes andproduces light and two carbonyl-containing compounds, one of which is anoxyanion-substituted ester compound containing two carboxylate groups,as shown below.

When M is H it is recognized that the respective dioxetane compound willpreferably only be used under conditions of pH where the carboxylic acidfunctions are ionized, i.e. pH> about 7. Preferably M is an alkali metalion, most preferably a sodium ion.

The groups R₃ and R₄ in another embodiment are combined together in acyclic or polycyclic alkyl group R₅ which is spiro-fused to thedioxetane ring, containing 6 to 30 carbon atoms which provides thermalstability and which can include additional non-hydrogen substituents.

The group R₅ is more preferably a polycyclic group, preferably anadamantyl group or a substituted adamantyl group having one or moresubstituent groups R₆ selected from halogens, alkyl, substituted alkyl,alkoxy, substituted alkoxy, carbonyl, carboxyl, phenyl, substitutedphenyl, amino and alkylamino groups covalently bonded thereto.

In another preferred embodiment the group R₂ is a phenyl or naphthylgroup. It is especially preferred that R₂ is a phenyl group in which theOX group is oriented meta to the dioxetane ring group as shown below.The phenyl ring may contain additional ring substituents R₇independently selected from halogens, alkyl, substituted alkyl, alkoxy,substituted alkoxy, carbonyl, carboxyl, amino and alkylamino groups.Some exemplary structures include by way of illustration:

Compounds of the latter two structural formulae in which R₆ is H or Cland R7 is Cl as shown below are recognized as further preferredcompounds.

The nature of the OX group is dictated by the triggering agent used inthe assay for which it is to be used and may be selected from hydroxyl,O⁻M⁺ wherein M is selected from hydrogen, an alkali metal ion or aquaternary ammonium or phosphonium ion, OOCR₈ wherein R₈ is selectedfrom the group consisting of alkyl and aryl groups containing 1 to 8carbon atoms and optionally containing heteroatoms, OPO₃ ⁻² salt, OSO₃ ⁻salt, β-D-galactosidoxy and β-D-glucuronidyloxy groups. The OX group ispreferably a OPO₃ ⁻² salt group.

Dioxetanes of the present invention having the formula:

wherein R₂, R₃, R₄, M and Z are as described above can be prepared usingmethods described in Applicants' co-pending application Ser. No.08/748,107 and other methods known in the art of dioxetane chemistry.For example, a ketone and ester having the formulas below wherein RG isa replaceable atom or group and X′ is a replaceable atom or group suchas a hydrogen or an alkyl group or a trialkylsilyl group can be coupledby a low-valent titanium reagent to form an intermediate vinyl ether.Removable groups include leaving groups such as halogen atoms selectedfrom Cl, Br and I, sulfates, sulfonates such as tosylate, mesylate andtriflate, quaternary ammonium groups, and azide.

The intermediate vinyl ether is converted in a process of one or moresteps to a precursor vinyl ether phosphate salt. It may be desired forsynthetic convenience to replace one removable group with anotherremovable group. The group RG is replaced by a CZ(COOM)₂ fragment byreaction with a Z-substituted malonate ester and later saponification ofthe ester groups. The group X′ is converted to the group X in the casewhere X and X′ are not identical by removing X′ and reacting with areagent which adds the X group or a protected form of the X group. Forexample when X′ is H and X is PO₃Na₂, treatment with base to deprotonatefollowed by reaction with a phosphorylating agent produces a phosphatetriester-protected vinyl ether which is converted to the phosphate saltby hydrolysis of the triester to the disodium salt. In this multi-stepprocess, two or more operations may occur in the same process step, forexample hydrolysis of carboxylic esters and phosphate esters can beeffected in the same step.

The precursor vinyl ether phosphate salt is directly converted to thedioxetane by known reactions including, for example, addition of singletoxygen generated by dye sensitization.

Each of these processes is exemplified by way of illustration in thespecific examples below. In particular, Scheme 1 depicts schematically asynthetic pathway used to prepare dioxetanes 3-5 according to the stepsdescribed above as disclosed and embodied in the aforementioned 748,107application.

Scheme 1

A preferred embodiment of the present invention concerns a process forpreparing a dioxetane salt compound of the formula IV:

having increased storage stability wherein R₃ and R₄ are each selectedfrom the group consisting of acyclic, cyclic and polycyclic organicgroups which can optionally be substituted with heteroatoms and whichcan optionally be joined together to form a cyclic or polycyclic ringgroup spiro-fused to the dioxetane ring, wherein R₂ is an aryl ringgroup selected from the group consisting of phenyl and naphthyl groupswhich can include additional substituents, wherein Z is selected fromthe group consisting of halogen atoms and alkyl groups of 1-4 carbonsand M is selected from hydrogen, an alkali metal ion or a quaternaryammonium or phosphonium ion comprising the steps of:

a) reacting a first alkene compound having the formula:

 wherein RG is a removable group with a Z-substituted malonate ester anda base to produce a malonate-substituted alkene compound having theformula:

 wherein R′ is an alkyl group of 1-4 carbons;

b) reacting the malonate-substituted alkene with a phosphorylatingreagent having the formula WP(O)Y₂ wherein W and Y are each halogenatoms, to form a phosphorylated alkene compound having the formula:

c) reacting the phosphorylated alkene compound with a hydroxyl compoundof the formula Y′—OH, wherein Y′ is selected from substituted orunsubstituted alkyl groups to form a second phosphorylated alkenecompound having the formula:

d) hydrolyzing the second phosphorylated alkene compound in an aqueoussolvent with a base of the formula M—Q wherein Q is a basic anion toform an alkene salt compound having the formula:

e) photooxidizing the alkene salt compound by irradiating a sensitizerin the presence of oxygen and the alkene salt compound in aqueoussolution to form the dioxetane salt compound.

It is more preferred that this process is used to prepare a dioxetane inwhich R₃ and R₄ are combined together to form a cyclic or polycyclicring group R₅ spiro-fused to the dioxetane ring and the dioxetane saltcompound has the formula:

In other preferred processes, the group R₂ is a meta-phenyl group, Z isa halogen or an alkyl group having 1-4 carbons atoms, more preferably Zis F or CH₃. and M is an alkali metal ion, more preferably M is Na.

It has now been discovered that compounds of formula IV

having increased storage stability wherein R₃ and R₄ are each selectedfrom the group consisting of acyclic, cyclic and polycyclic organicgroups which can optionally be substituted with heteroatoms and whichcan optionally be joined together to form a cyclic or polycyclic ringgroup spiro-fused to the dioxetane ring, wherein R₂ is an aryl ringgroup selected from the group consisting of phenyl and naphthyl groupswhich can include additional substituents, wherein z is selected fromthe group consisting of halogen atoms and alkyl groups of 1-4carbons,and M is selected from hydrogen, an alkali metal ion or aquaternary ammonium or phosphonium ion can be advantageously prepared byan improved process comprising the steps of:

a) reacting a first alkene compound having the formula:

 wherein RG is a removable group with a Z-substituted malonate ester anda base to produce a malonate-substituted alkene compound having theformula:

 wherein R′ is an alkyl group of 1-4 carbons;

b) photooxygenating the malonate-substituted alkene compound byirradiating a sensitizer in the presence of oxygen and themalonate-substituted alkene compound to form a malonate-substituteddioxetane having the formula:

c) reacting the malonate-substituted dioxetane with a phosphorylatingreagent having the formula WP(O)Y₂ wherein W is selected from halogensand Y is selected from halogen atoms, substituted or unsubstitutedalkoxy, aryloxy, aralkyloxy and trialkylsilyloxy groups to form aphosphorylated dioxetane compound having the formula:

d) hydrolyzing the phosphorylated dioxetane in an aqueous solvent with abase of the formula M—Q wherein Q is a basic anion to form the dioxetanesalt compound.

It is more preferred that this process is used to prepare a dioxetane inwhich R₃ and R₄ are combined together to form a cyclic or polycyclicring group R₅ spiro-fused to the dioxetane ring and which can containadditional substituents and the dioxetane salt compound has the formula:

In other preferred embodiments, the process is used to prepare adioxetane in which the group R₂ is a meta-phenyl group which can containadditional substituents, Z is a halogen or an alkyl group having 1-4carbons atoms, more preferably Z is F or CH₃, and M is an alkali metaliion, more preferably M is Na.

The step of reacting the first alkene compound with the Z-substitutedmalonate ester CHZ(COOR′)₂ and a base to produce a malonate-substitutedalkene compound is generally performed in a polar aprotic solvent suchas DMSO, DMF, N,N-dimethylacetamide, N-methylpyrollidone using a poorlynucleophilic base, preferably sodium or potassium hydride. The reactionis preferably performed at an elevated temperature to decrease reactiontime, generally between 50 and 150° C., more usually between 80 and 120°C. Removable groups include leaving groups such as halogen atomsselected from Cl, Br and I, sulfates, sulfonates such as tosylate,mesylate and triflate, quaternary ammonium groups, and azide.

In the improved process described herein, the photooxygneation step isperformed on the intermediate malonate-substituted alkene instead ofphotooxygenating a phosphate alkene as the final step of the overallprocess as described in the 748,107 application. In this step, themalonate-substituted alkene compound bearing a phenol group is dissolvedin an organic solvent and irradiated in the presence of a sensitizer andoxygen to form a malonate-substituted dioxetane. Irradiation of asensitizer and oxygen with light, usually visible light, generatessinglet oxygen which reacts with the vinyl ether-type double bond of themalonate-substituted alkene. The sensitizer is can be dissolved in thesolvent or, preferably, immobilized on a polymeric particle as iscommonly known in the art. Sensitizers useful for generating singletoxygen include, without limitation, Rose Bengal, methylene blue, eosin,tetraphenylporphyrin (TPP) metal complexes of TPP, especially zinc andmanganese and C₆₀. Preferred organic solvents include halocarbons suchas CH₂C₂, CHCl₃ and CCl₄, deuterated halocarbons, low molecular weightketones and their deuterated analogs, aliphatic and aromatichydrocarbons and their deuterated analogs. Most preferred is CH₂Cl₂.Conducting the photooxygenation in an organic solvent advantageouslyprovides a reaction medium in which the lifetime of singlet oxygen ismaximized. This has the effect of significantly decreasing reactiontimes and permitting the photooxygenation to proceed more readily tocompletion. Product isolation is facilitated as well, in most casesrequiring only a simple filtration of sensitizer and evaporation ofsolvent.

The step of reacting the malonate-substituted dioxetane with aphosphorylating reagent having the formula WP(O)Y₂ wherein W is selectedfrom halogens and Y is selected from halogen atoms, substituted orunsubstituted alkyloxy groups and trialkylsilyloxy groups to form aphosphorylated dioxetane compound is performed in an organic solvent,preferably a halocarbon such as CH₂Cl₂ or CHCl₃ or an ether such asdiethyl ether or tetrahydrofuran (THF) in the presence of an amine base.Useful amine bases include, without limitation, pyridine andtriethylamine. When Y is a substituted or unsubstituted alkyloxy group,an aryloxy, aralkyloxy or trialkylsilyloxy group, representative Ygroups include, by way of example, alkoxy such as OCH₃, OCH₂CH₃. and thelike, substituted alkoxy such as cyanoethoxy (OCH₂CH₂CN) ortrimethylsilylethoxy (OCH₂CH₂Si(CH₃)₃), phenoxy, substituted phenoxy,benzyloxy, trimethylsilyloxy and others as are generally known to theskilled organic chemist. The two groups Y can also be combined togetheras a single group such as ethylenedioxy as occurs in the reagent

Preferred groups Y are cyanoethoxy groups. In a more preferredembodiment, Y is a halogen, preferably Y and W are both Cl.

The phosphorylation step is performed in solution at a temperature inthe range of about −78° C. to about 25° C. A temperature of about 0-5°C. is particularly convenient. The phosphorylating agent WP(O)Y₂ isadded in a controlled fashion so as not to cause the reaction solutionto become hot. The phosphorylating reagent is preferably accompanied byan amine base during the addition, preferably pyridine.

The hydrolysis or deprotection step is accomplished by hydrolyzing thephosphorylated dioxetane in an aqueous solvent with a base of theformula M—Q wherein Q is a basic anion in a quantity sufficient to causeremoval of the protecting groups Y and R′ to form the dioxetane saltcompound. The solvent can comprise water, an aqueous buffer or a mixtureof water and one or more organic solvents. Preferred orgnaic solventsare water-miscible solvents such as methanol, ethanol, acetone and THF.Four equivalents of the base are typically required, however forconvenience, an excess can be employed. Removal of the protecting groupscan be performed sequentially or simultaneously. Depending on theparticular groups Y and R′ and the base it may or may not be possible toisolate partially hydrolyzed intermediates.

The choice of the basic deprotecting agent will be determined, in part,by the nature of the groups Y and R′ to be removed. The deprotectingagent must also not cause undesired side reactions such as hydrolysis ofthe vinyl ether group in the process where the vinyl ether phosphatesalt is first prepared or decomposition of the dioxetane ring group inthe process where the protected dioxetane is prepared. Preferreddeprotecting agents include organic and inorganic bases such as sodiumhydroxide, potassium hydroxide, potassium carbonate, sodium methoxide,sodium ethoxide, potassium t-butoxide, ammonia, ammonium hydroxide andthe like. Other preferred deprotecting agents include nucleophilicagents such as cyanide ion, fluoride ion.

In another embodiment, the step of reacting the malonate-substituteddioxetane compound with the phosphorylating reagent comprises the stepsof:

a) reacting the malonate-substituted dioxetane compound with aphosphorylating reagent having the formula WP(O)Y′₂ wherein W and Y′ areeach halogen atoms to form a dioxetane phosphoryl halide compound havingthe formula

b) reacting the dioxetane phosphoryl halide compound with a hydroxylcompound of the formula Y—OH, wherein Y is selected from substituted orunsubstituted alkyl groups to form the phosphorylated dioxetanecompound.

The dioxetane phosphoryl halide compound is converted to thephosphorylated dioxetane compound by reaction with at least twoequivalents of a hydroxyl compound Y—OH and preferably with an excess.Exemplary compounds which can serve as the hydroxyl compound Y—OHinclude, without limitation, lower alcohols such as methanol andethanol, substituted lower alcohols such as 3-hydroxyprdpionitrile(HOCH₂CH₂CN) and 2-trimethylsilylethanol, phenol, substituted phenols,benzyl alcohol and others as are generally known.

In another aspect, the present invention relates to syntheticintermediates used in the processs for preparing the present dioxetanes.In particular the following novel alkene intermediate compounds areuseful.

Additionally the following novel dioxetane compounds are useful assynthetic intermediates in the preparation of the present dioxetanecompounds.

An exemplary synthesis of a dioxetane of the present invention by thisimproved process is shown in Scheme 2.

Scheme 2

The starting material (precursor alkene) in the above describedsynthetic processes having the formula:

wherein RG is a removable group can be prepared by methods known in theart. In one method, the vinyl ether function is prepared by Ti-mediatedcoupling of a ketone R₃R₄C═O and an ester HOR₂COOCH₂CH₂—G as describedin U.S. Pat. Nos. 4,983,779 and 4,982,192 wherein G is a group which maybe identical with RG or may be a group which can be replaced by RG orconverted into RG. An exemplary synthetic process in which RG is aniodine atom and G is a chlorine atom is presented hereinbelow. It isfurther recognized that, for convenience, the ester component of thecoupling reaction may be used in protected form in which the hydroxylgroup is present in a masked form such as a silyl ether or an alkylether. After the coupling reaction, the free hydroxyl group is thenliberated using standard synthetic means.

It has further been discovered by Applicants that these precuror alkenescan also be prepared by a new process not previously reported for thepreparation of this type of vinyl ether. While the foregoing Ti-mediatedprocess requires the preparation of individual ester compounds bearingthe G or RG group, adding additional complexity and cost, the newprocess utilizes a common vinyl ether intermediate which can be preparedfrom commercially available starting materials.

An example of a reaction for preparing the precursor alkene by the newprocess is depicted below. A lower alkyl vinyl ether compound, whereinlower alkyl, R₉, here indicates a C₁-C₄ straight or branched alkylgroup, is reacted with a catalytic amount of a mercury salt in thepresence of at least one mole equivalent of another alcohol R₁₀—OH, e.g.one having the formula HOCH₂CH₂G, to produce the desired precursoralkene.

The conversion of unsubstituted vinyl ethers having the formulaCH₂═CHOR_(a) to other unsubstituted vinyl ethers having the formulaCH₂═CHOR_(b) is known and described, e.g. in W. H. Watanabe and L. E.Conlon, J. Am. Chem.Soc. 79, 2828 (1957), the preparation by a mercurysalt-catalyzed reaction of trisubstituted alkenes used in the presentprocesses has not been reported to the best of Applicants' knowledge.

In this reaction process, R₃ and R₄ are each selected from acyclic,cyclic and polycyclic organic groups which can optionally be substitutedwith heteroatoms and which can optionally be joined together to form acyclic or polycyclic ring group R₅ spiro-fused to the dioxetane ring, R₂is an aryl ring group selected from phenyl and naphthyl groups which caninclude additional substituents. An example of the use of thismercury-catalyzed reaction for the preparation of an alkene precursor toa dioxetane of the invention is

wherein G is a chlorine atom. The mercury salt is any Hg(II) salt whichfunctions to catalyze the vinyl ether groups and is preferably a salt ofa weak acid such as acetate or trifluoroacetate. The mercury salt isused in catalytic quantitity, typically from 0.01 to 0.5 moles per moleof alkene, more typically from 0.05 to 0.25. The alcohol componentR₁₀—OH can be any alkanol, substituted alkanol, benzyl alcohol,unsaturated alcohol, such as allyl alcohol. The alcohol is used inexcess, at least two moles per mole of alkene and preferably at least 5moles per mole of alkene. In a preferred process, the alcohol is used asthe reaction solvent. The reaction is typically but not necessarilyconducted above ambient temperature up to the boiling point of thesolvent. Preferable reaction temperatures are in the range of about70-120° C. Additional solvents for purposes of improving the solubilityof reactants or altering polarity or boiling point can be used.

It is recognized that while the mercury-catalyzed vinyl ether exchangereaction described above will find particular use in the preparation ofintermediates used for the further elaboration to water solubletri-substituted dioxetane of the present invention, it is more generallyapplicable to the preparation of a wide variety of alkene or vinyl ethercompounds.

Specific Embodiments

A fluoro-substituted analog of dioxetane 1, identified as 3, achloro-substituted analog 4 and a methyl-substituted analog 5 have beenprepared and their storage stability evaluated over several weeks.Storage stability of a solution of 1 was measured for comparison. Allsolutions were prepared with the same composition, differing only in theidentity of the dioxetane. Stability was evaluated by chemiluminescentenzyme assay with a fixed volume of test solution and fixed limitingamount of alkaline phosphatase and measuring the plateau light intensityat 25° C. Unexpectedly, aqueous solutions containing dioxetanes 3 and 5were substantially more stable than 1, while dioxetane 4 was not.Solutions of dioxetanes 3 or 5 underwent essentially no decompositionafter four weeks at 25° C. Surprisingly, the storage stability ofdioxetane 4 was actually worse than that of 1.

The reasons for this difference in the properties of these fourdioxetanes are not presently understood. It is particularly significantthat dioxetanes 3 and 4 should show such marked difference in storagestability when they differ structurally only by having different halogensubstituents. Applicants are aware of no teachings in the art ofdioxetane chemistry to explain or predict these results.

Furthermore, tests on dioxetane 3, showed that, like dioxetane 1, itcaused no carryover in the capsule chemistry assay system. Dioxetanessuch as 3 and 5 bearing a substituent containing two carboxylate groupsand either a fluorine atom or a lower alkyl group and compositionscontaining such dioxetanes are therefore superior to other knowndioxetanes and compositions for use in capsule chemistry analysissystems.

In another aspect of the invention, compositions providing enhancedchemiluminescence are provided. Enhanced compositions are advantageousin assays requiring the highest analytical sensitivity. Increasing thechemilumin-escence efficiency of the dioxetane decomposition reactionwhile maintaining or reducing extraneous light emission from spontaneousdioxetane decomposition is one manner in which sensitivity can beenhanced or improved.

The present invention, therefore, also relates to compositionscomprising a cationic enhancer and a stable 1,2-dioxetane as describedabove having increased storage stability which can be triggered togenerate chemiluminescence. Such compositions for providing enhancedchemiluminescence comprise a dioxetane as described above in an aqueoussolution, and a non-polymeric cationic enhancer substance whichincreases the quantity of light produced by reacting the dioxetane withthe activating agent compared to the amount which is produced in theabsence of the enhancer. It is preferred that the enhancer substance isa dicationic surfactant of the formula:

wherein each of A is independently selected from P and N atoms andwherein Link is an organic linking group containing at least two carbonatoms selected from the group consisting of substituted andunsubstituted aryl, alkyl, alkenyl and alkynyl groups and wherein Linkmay contain heteroatoms and wherein R is selected from lower alkyl oraralkyl containing 1 to 20 carbon atoms and wherein Y is an anion. It isespecially preferred that the enhancer substance is a dicationicsurfactant having the formula:

and wherein link is phenylene.

Compositions of the present invention for providing enhancedchemiluminescence may optionally contain at least one fluorescer as asupplementary enhancer. Fluorescers useful are those compounds which arecapable of increasing the quantity of light produced through energytransfer. Anionic fluorescers are particularly effective it is believeddue to favorable electrostatic interactions with the cationic enhancer.Particularly preferred fluorescers are anionic compounds and include,without limitation, pyranine and fluorescein.

In order to more fully describe the various aspects of the presentinvention, the following non-limiting examples describing particularembodiments are presented for purposes of illustration of the invention.

EXAMPLES Example 1 Preparation of Dioxetane 1

The dioxetane[4-(3,3-biscarboxy)propoxy)-4-(3-phosphoryloxyphenyl)]spiro[1,2-dioxetane-3,2′-tricyclo-[3.3.1.1^(3,7)]decane],tetrasodium salt was prepared by the sequence of reactions described inApplicants' U.S. Pat. No. 5,631,167. The synthesis up to theintermediate alkene[(3-hydroxyphenyl)-(2-iodoethoxy)-methylene]tricyclo(3.3.1.1^(3,7)]decanewas conducted essentially as described in U.S. Pat. Nos. 5,013,827 and5,068,339.

Example 2 Preparation of Dioxetane 2

The dioxetane[4-(3-carboxypropoxy)-4-(3-phosphoryloxyphenyl)]spiro[1,2-dioxetane-3,2′-tricyclo[3.3.1.1^(3,7)]-decane](2) was prepared by the sequence of reactions described in Applicants'U.S. Pat. No. 5,631,167. The synthesis up to the intermediate alkene[(3-carboxypropoxy)-(3-hydroxyphenyl)methylene]-tricyclo-[3.3.1.1^(3,7)]decanewas conducted essentially as described in U.S. Pat. Nos. 5,013,827 and5,068,339.

Example 3 Preparation of Dioxetane 3

This dioxetane was prepared by the sequence of reactions describedbelow. The synthesis up to the intermediate alkene[(3-hydroxyphenyl)-(2-iodoethoxy)methylene]tricyclo[3.3.1.1^(3,7)]decanewas conducted as described in Example 1.

(a) Synthesis of[((3,3-biscarboethoxy)-3-fluoropropoxy)-(3-hydroxyphenyl)methylenetricyclo[3.3.1.1^(3,7)]decane.Sodium hydride (75 mg of a 60% dispersion in oil) was washed free of oilwith hexane, dried under vacuum and added to 4 mL of anhydrous DMSO.Diethyl fluoromalonate (0.3 g) was added and the suspension stirredunder Ar for 15 min. A solution of the iodoethoxy alkene (0.5 g) in 5 mLof anhydrous DMSO was added to the reaction mixture. The reaction washeated to 100° C. and stirred for 2 h. After cooling, the mixture wasdiluted with 30 mL of ethyl acetate. The ethyl acetate solution wasextracted 3-4 times with water, dried and evaporated. The crude materialwas chromatographed using 5-20% ethyl acetate in hexane. The desiredcompound (0.25 g) was obtained in 45% yield: ¹H NMR (CDCl₃) δ 1.28(t,6H), 1.66-1.95 (m,12H), 2.45 (t,1H), 2.52 (t,1H), 2.67(br s,1H), 3.20(br s,1H), 3.52(t,2H), 4.23-4.30 (q,4H), 6.74-7.22 (m,4H).

(b) Synthesis of[((3,3-biscarboethoxy)-3-fluoropropoxy-(3-(bis-(2-cyanoethyl)phosphoryloxy)phenyl)methylene]tricyclo[3.3.1.1^(3,7)]decane.A flask containing 10 mL of CH₂Cl₂ under a layer of argon was cooled inan ice bath. Pyridine (1.71 mL) was added followed by slow addition ofPOCl₃ (0.61 mL) and stirring continued for 15 min. A solution of thealkene (0.972 g) from step (a) in 10 mL of CH₂Cl₂ was added dropwise.The ice bath was removed and the solution stirred for 2.5 h. To thissolution was added 1.71 mL of pyridine and 1.44 mL of 2-cyanoethanol.The reaction mixture was stirred for 12-15 h resulting in formation of awhite precipitate. The mixture was diluted with CH₂Cl₂ and washed with4×50 mL of water. The CH₂Cl₂ extract was dried and evaporated. The crudeproduct was purified by chromatography using 75% ethyl acetate inhexane. A total of 1.2 g of an oil (88%) was obtained: ¹H NMR (CDCl₃) δ1.29 (s,6H), 1.79-1.97 (m,12H), 2.46-2.53 (2t,2H), 2.63 (br s,1H), 2.83(t,4H), 3.20 (br s,1H), 3.50 (t,2H), 4.24-4.31 (q,4H), 4.35-4.51 (m,4H),7.13-7.36 (m,4H); ³¹P NMR (CDCl₃) δ −9.49 (p).

(c) Synthesis of[(3,3-biscarboxy-3-fluoropropoxy)-(3-phosphoryloxyphenyl)methylene]tricyclo[3.3.1.1^(3,7)]-decane,tetrasodium salt. The alkene (1.2 g) from step (b) was dissolved in 20mL of acetone. A solution of 297 mg of sodium hydroxide in 4 mL of waterwas added. The solution was stirred over night during which time aprecipitate formed. The liquid was decanted and the solid washed with10×5 mL of acetone. After drying under vacuum, a white solid (1.0 g) wasobtained: ¹H NMR (D₂O) δ 1.75-1.89 (m,12H), 2.29 (t,2H), 2.37 (t,2H),2.57 (br s,1H), 3.12 (br s,1H), 3.56 (t,2H), 6.99-7.30 (m,4H); ³¹P NMR(D₂O) δ 0.69 (s).

(d) Synthesis of[4-(3,3-biscarboxy)-3-fluoropropoxy)-4-(3-phosphoryloxyphenyl)]spiro[1,2-dioxetane-3,2′-tricyclo[3.3.1.1^(3,7)]decane], tetrasodium salt (3). The alkene (348.6 mg) fromstep (c) was dissolved in 10 mL of D₂O. Polymer-bound Rose Bengal (500mg) was suspended in 10 mL of p-dioxane and added to the water solution.The reaction mixture was cooled to 5-8° C., oxygen bubbling was startedand the mixture irradiated with a sodium lamp through a 5 mil KAPTONfilter. After a total of 2.5 h, the polymer beads were filtered off andthe solution was evaporated to dryness producing a white solid (3). ¹HNMR (D₂O) δ 0.93-1.79 (m,12H), 2.19 (br s,1H), 2.41-2.49 (m,2H), 2.97(br s,1H), 3.40-3.49 (m,2H), 7.19-7.42 (m,4H); ³¹P NMR (D₂O) δ 0.575(s).

Example 4 Preparation of Dioxetane 4

This dioxetane was prepared by the sequence of reactions describedbelow. The synthesis up to the intermediate alkene[(3-hydroxyphenyl)-(3,3-biscarboethoxy)propoxymethylene]tricyclo[3.3.1.1^(3,7)]decanewas conducted as described in Example 1.

(a) Synthesis of[((3,3-biscarboethoxy)-3-chloropropoxy)-(3-hydroxyphenyl)methylenetricyclo[3.3.1.1^(3,7)]decane.A solution of(3,3-biscarboethoxypropoxy)-(3-hydroxyphenyl)methylenetricyclo[3.3.1.1^(3,7)]decane(1.2 g) in 10 mL of dry THF was added to a 2.4 eq. of LDA in 25-30 mL ofdry THF at −78° C. under argon. The reaction was stirred for 30 min at−78° C. and treated with a solution of N-chloro-succinimide (0.58 g) in15 mL of dry THF. The reaction was allowed to warm to room temperatureover an hour and stirred for an additional hour. The THF was removed invacuo and the residue dissolved in 100 mL of ethyl acetate. The organicsolution was washed with water, dried and evaporated. The crude materialwas separated by column chromatography. ¹H NMR (CDCl₃) δ 1.23 (t,6H),1.7-2.00 (m,12H), 2.57 (t,2H), 2.65 (br s,1H), 3.2 (br s,1H), 3.56(t,2H), 4.22 (q,4H), 6.65-7.25 (m,4H).

(b) Synthesis of[((3,3-biscarboethoxy)-3-chloropropoxy-(3-(bis-(2-cyanoethyl)phosphoryloxy)phenyl)methylene]tricyclo[3.3.1.1^(3,7)]decane.A flask containing 25 rnL of CH₂Cl₂ under a layer of argon was cooled inan ice bath. Pyridine (1.5 g) was added followed by slow addition ofPOCl₃ (1.82 g) and stirring continued for 15 min. A solution of thealkene (1.5 g) from step (a) and 1.5 g of pyridine in 25 mL of CH₂Cl₂was added dropwise. The ice bath was then removed and the solutionstirred for 1 h. The solution was again cooled with an ice bath andtreated sequentially with 3.0 g of pyridine and 2.8 g of 2-cyanoethanol.The reaction mixture was stirred for 12-15 h resulting in formation of awhite precipitate. The mixture was diluted with CH₂Cl₂ and washed withwater. The CH₂Cl₂ extract was dried and evaporated. The crude productwas purified by chromatography using 50% ethyl acetate in hexane. Atotal of 1.4 g of product was obtained an oil: ¹H NMR (CDCl₃) δ 1.278(t,6H), 1.80-1.97 (m,12H), 2.565 (t,2H), 2.63 (br s,1H), 2.826 (t,4H),3.20 (br s,1H), 3.556 (t,2H), 4.271 (q,4H), 4.40-4.47 (m,4H), 7.15-7.36(m,4H).

(c) Synthesis of[(3,3-biscarboxy-3-chloropropoxy)-(3-phosphoryloxyphenyl)methylene]tricyclo[3.3.1.1^(3,7)]-decane,tetrasodium salt. The alkene (0.9 g) from step (b) was dissolved in 25mL of acetone. A solution of 0.22 g of sodium hydroxide in 3 mL of waterwas added. The solution was stirred over night during which time aprecipitate formed. The liquid was decanted and the solid trituratedwith acetone. The white solid was filtered, washed further with acetoneand dried under vacuum: ¹H NMR (D₂O) δ 1.77-1.92 (m,12H), 2.422 (t,2H),2.59 (br s,1H), 3.15 (br s,1H), 3.635 (t,2H), 7.02-7.33 (m,4H).

(d) Synthesis of[4-(3,3-biscarboxy)-3-chloropropoxy)-4-(3-phosphoryloxyphenyl)]spiro[1,2-dioxetane-3,2′-tricyclo[3.3.1.1^(3,7)]decane], tetrasodium salt (4). The alkene (35 mg) fromstep (c) was dissolved in 1.0 mL of D₂O. Polymer-bound Rose Bengal (500mg) was soaked in 1.0 mL of p-dioxane-d₈ for 5 min and then added to thewater solution. The reaction mixture was cooled to 0° C., oxygenbubbling was started and the mixture irradiated with a sodium lampthrough a 5 mil KAPTON filter for 45 min to produce 4 as determined byNMR. The mixture was filtered and the solution diluted in buffer forenzyme assay: ¹H NMR (D₂O) δ 1.05-1.96 (m, 12H), 2.19 (br s,1H),2.60-2.62 (m,2H), 3.07 (br s,1H), 3.56-3.58 (m,2H), 7.25-7.44 (m,4H).

Example 5 Preparation of Dioxetane 5

This dioxetane was prepared by the sequence of reactions describedbelow. The synthesis up to the intermediate alkene[(3-hydroxyphenyl)-(2-iodoethoxy)methylene]tricyclo-[3.3.1.1^(3,7)]decanewas conducted as described in Example 1.

(a) Synthesis of[((3,3-biscarboethoxybutoxy)-(3-hydroxyphenyl)methylenetricyclo[3.3.1.1^(3,7)]decane.Sodium hydride (0.866 g of a 60% dispersion in oil) was washed free ofoil with hexane, dried under vacuum and added to 15 mL of anhydrousDMSO. Diethyl methylmalonate (2.4 g) was added and the suspensionstirred under Ar for 15 min. A solution of the iodoethoxy alkene (2.8 g)in 15 mL of anhydrous DMSO was added to the reaction mixture. Thereaction was heated to 100° C. and stirred for 2 h. After cooling, themixture was diluted with 30 mL of ethyl acetate. The ethyl acetatesolution was extracted 3-4 times with water, dried and evaporated. Thecrude material was chromatographed using 5-20% ethyl acetate in hexane.The desired compound (0.80 g) was obtained in 25% yield: ¹H NMR (CDCl₃)δ 1.208 (t,6H), 1.347 (s,3H), 1.76-1.96 (m,12H), 2.20 (t,2H), 2.66 (brs,1H), 3.20 (br s,1H), 3.41 (t, 2H), 4.09-4.17 (q,4H), 6.78-7.26 (m,4H).

(b) Synthesis of[((3,3-biscarboethoxybutoxy-3-(bis-(2-cyanoethyl)phosphoryloxy)phenyl)methylene]tricyclo-[3.3.1.1^(3,7)]decane.A flask containing 15 mL of CH₂Cl₂ under a layer of argon was cooled inan ice bath. Pyridine (1.38 g) was added followed by slow addition ofPOCl₃ (0.8 g) and stirring continued for 15 min. A solution of thealkene (0.8 g) from step (a) in 15 mL of CH₂Cl₁ was added dropwise. Theice bath was removed and the solution stirred for 1 h. To this solutionwas added 1.38 g of pyridine and 1.24 g of 2-cyanoethanol. The reactionmixture was stirred for 12-15 h resulting in formation of a whiteprecipitate. The mixture was diluted with CH Cl and washed with 4×50 mLof water. The CH₂Cl₂ extract was dried and evaporated. The crude productwas purified by chromatography using 75% ethyl acetate in hexane. Atotal of 0.55 g of an oil (50%) was obtained: ¹H NMR (CDCl₃) δ 1.208(t,6H), 1.34 (s,3H), 1.78-1.97 (m,12H), 2.18 (t,2H), 2.61 (br s,1H),2.81 (t,4H), 3.21 (br s,1H), 3.41 (t,2H), 4.09-4.16 (q,4H), 4.37-4.46(m,4H), 7.14-7.34 (m,4H).

(c) Synthesis of[(3,3-biscarboxybutoxy)-(3-phosphoryloxyphenyl)methylene]tricyclo(3.3.1.1^(3,7)]decane,tetrasodium salt. The alkene (0.47 g) from step (b) was dissolved in 14mL of acetone. A solution of 0.117 g of NaOH in 1.5 mL of water wasadded. The solution was stirred over night during which time aprecipitate formed. The liquid was decanted and the solid washed with10×5 mL of acetone. After drying under vacuum, a white solid (0.383 g,92%) was obtained: ¹H NMR (D₂O) δ 1.09 (s,3H), 1.75-1.90 (m,12H), 2.00(t,2H), 2.57 (br s,1H), 3.13 (br s,1H), 3.47 (t,2H), 7.01-7.29 (m,4H).

(d) Synthesis of[4-(3,3-biscarboxybutoxy)-4-(3-phosphoryloxyphenyl)]spiro[1,2-dioxetane-3,2′-tricyclo[3.3.1.1^(3,7)]-decane],tetrasodium salt (5). The alkene (65 mg) from step (c) was dissolved in3 mL of D₂O. Polymer-bound Rose Bengal (35 mg) was suspended in 3 mL ofp-dioxane and added to the water solution. The reaction mixture wascooled to 5-8° C., oxygen bubbling was started and the mixtureirradiated with a sodium lamp through a 5 mil KAPTON filter for 1 h toproduce (5). The polymer beads were filtered off and the solution usedfor preparing stock solutions for testing. ¹H NMR (D₂O) δ 0.92-1.33 (m,5H), 1.38-2.21 (m, 13H) , 2.92 (br s,1H), 3.19-3.32 (m,2H), 7.14-7.73(m,4H).

Example 6 Alternative Preparation of Dioxetane 3

The dioxetane was prepared by the sequence of reactions described belowusing [(3-hydroxyphenyl)methoxymethylene-tricyclo[3.3.1.1^(3,7)]decaneas starting material. This compound can be prepared as described in U.S.Pat. No. 4,983,779.

(a) The alkene[(3-hydroxyphenyl)methoxymethylene-tricyclo[3.3.1.1^(3,7)]decane (12 g)was added to 100 mL of 2-chloroethanol and stirred. A catalytic amountof Hg(OAc)₂ (2.8 g) was then added to the mixture under an argonatmosphere. The reaction was stirred for 5 h at 110° C. After cooling toroom temperature, the chloroethanol was remove under vacuum. The solidwas dissolved in EtOAc and washed with water. The EtOAc layer was driedover Na₂SO₄ and evaporated to produce[(3-hydroxyphenyl)-(2-chloroethoxy)methylene]tricyclo[3.3.1.1^(3,7)]decane.

(b) Replacement of the chlorine atom in the above compound with aniodine atom was conducted essentially as described in U.S. Pat. Nos.5,013,827 and 5,068,339.

(c) Synthesis of[(3-hydroxyphenyl)-(3,3-biscarboethoxy)-3-fluoropropoxymethylene]tricyclo[3.3.1.1^(3,7)]decanefrom[(3-hydroxyphenyl)-(2-iodoethoxy)methylene]tricyclo-[3.3.1.1^(3,7)]decaneis described in Example 3 above.

(d) The fluoromalonate alkene from step (c) (0.375 g) wasphotooxygenated with ca. 1 mg of methylene blue in 15 mL of CH₂Cl₂.After cooling the solution to −78° C. with O₂ bubbling, the solution wasirradiated with a sodium lamp through a 5 mil KAPTON filter for 45 minand then allowed to warm to room temperature. The CH₂Cl₂ was evaporatedand the residue chromatographed using from 0-5% EtAc in CH₂Cl₂ as eluentto produce(4-(3,3-biscarboethoxy-3-fluoropropoxy)-4-(3-hydroxyphenyl)]spiro[1,2-dioxetane-3,2′-tricyclo[3.3.1.1^(3,7)]-decane]:¹H NMR (CDCl₃) δ 0.97-1.02 (m,1H), 1.21-1.33 (m,7H), 1.45-1.91 (m,10H),2.23 (br s,1H), 2.48-2.80 (m,2H), 2.96 (br s,1H), 3.35-3.44 (m,1H),3.65-3.75 (m,1H), 4.21-4.40 (m,4H), 6.85-7.40 (m,4H).

(e) The dioxetane from the previous step was phosphorylated by thefollowing process. A solution of 2 mL of anhydrous pyridine and 10 mL ofCH₂Cl₂ under argon was cooled to 0° C. and a solution of 0.424 g ofPOCl₃ in 10 mL of CH₂Cl₂ was added dropwise. After 15 min, a solution of0.424 g of the dioxetane in 10 mL of CH₂Cl₂ was added dropwise. Thesolution was allowed to warm to room temperature and stirred for 4 h.The solution was again cooled to 0° C. and a solution of 0.75 g ofcyanoethanol in 10 mnL of CH₂Cl₂ was added dropwise. This solution wasallowed to warm to room temperature as it was stirred for 2.5 h. Afterevaporating to dryness, the residue was chromatographed using from50-100% ethyl acetate in hexanes as eluent. The solvents were thenremoved in vacuo yielding a colorless oil. The dioxetane was thendissolved in 100 inL of CH₂Cl₂ and washed three times with type I water.The organic layer was then dried over Na₂SO₄. filtered, and evaporatedto produce the phosphorylated dioxetane : ¹H NMR (CDCl₃) δ 0.90-0.95(m,1H), 1.24-1.33 (m,7H), 1.46-2.20 (m,11H), 2.50-2.86 (m,6H), 2.96 (brs,1H), 3.32-3.41 (m,1H), 3.62-3.73 (m,1H), 4.20-4.48 (m,8H), 7.30-7.70(m,4H); ³¹P (CDCl₃) −9.53 (p).

(f) the alkyl groups were removed by reacting the dioxetane from theprevious step with 47.2 mg of NaOH in 1 mL of type I water and 10 mL ofacetone under argon over night. Solvent was decanted from the oilyresidue which had formed. The oil was then washed twice with 2 mL ofacetone and then triturated with another 10 mL of acetone to produce apowdery white solid. Solid dioxetane 3 was collected by suctionfiltration and washed with another 20 mL of acetone.

In an alternative procedure, the dioxetane product of step (d) can bedirectly converted to dioxetane 3 by by the following process. Asolution of 2 mL of anhydrous pyridine and 10 mL of CH₂Cl2 under argonis cooled to 0° C. and a solution of 0.424 g of POCl₃ in 10 mL of CH₂Cl₂is added dropwise. After 15 min, a solution of 0.424 g of the dioxetanein 10 mL of CH₂Cl₂ is added dropwise. The solution is allowed to warm toroom temperature and stirred for 4 h. The phosphate salt is formed andthe ester groups are hydrolyzed by reacting the resultingdichlorophosphate dioxetane with 47.2 mg of NaOH in 1 mL of type I waterand 10 mL of acetone under argon over night. The solvent is removed fromthe residue containing the product. The product is then washed withacetone and, if needed, triturated with acetone to produce a powderywhite solid. Dioxetane 3 is collected by suction filtration.

Example 7 Discovery of Reagent Carrvover Problem in Capsule ChemistryAnalysis System

The experiments described below were performed on a prototype capsulechemistry analysis system essentially as described by Kumar et al inU.S. Pat. No. 5,399,497, with the detection system configured to measurelight emission (luminescence). The method and apparatus comprisesfeeding a stream of fluid segments through a Teflon tube, where the tubehas an isolating layer of fluorocarbon oil on the inner surface. Sampleand reagents are aspirated into this tube, and the resulting liquidsegments are moved through the tube. Separation steps and washing stepswhich are required by heterogeneous immunoassay methods were facilitatedby means of magnets, which transferred magnetic particles from oneaqueous segment to another. The detection system was comprised of aphoton counter and a fiber optic read head, in which the fibers wereradially arranged around the Teflon tube to maximize the efficiency oflight collection.

The TECHNICON IMMUNO 1® TSH method (Bayer Corporation, Tarrytown, N.Y.,USA) was used as a representative immunoassay method for the testing ofluminogenic reagents. The method principle involved incubation of aspecimen containing the antigen TSH with a first reagent (R1), whichcontained a fluorescein-labeled antibody, and simultaneously with asecond reagent (R2), which contained an antibody-alkaline phosphatase(ALP) conjugate. Each antibody was specific for a different epitope onthe TSH antigen, so that formation of a “sandwich” was promoted betweenthese two antibodies and the TSH antigen. Magnetic particles containingbound anti-fluorescein were used to capture the sandwich, and theparticles were subsequently washed to remove unbound reagents. Theparticles were then exposed to the luminogenic reagent, which containeda substrate for ALP, and luminescence was measured.

The luminogenic R3 reagent was comprised of 0.2 mM CSPD (disodium3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.1^(3,7)]decan}-4-yl)phenyl phosphate, (Tropix, Inc., Bedford,Mass., USA), 3 mM pyranine (hydroxypyrenesulfonic acid), 1 mM MgCl₂, 1 Mdiethanolamine buffer (pH 10.0), 0.1% Triton X-100 and 0.1% NaN₃. Thesequence of events on the capsule chemistry analysis system is depictedin FIG. 1 of the drawings. The fluid capsule or test package wascomprised of six liquid segments, each of which had a volume of 28 μl.Magnetic particles (1.4 μl of the magnetic particle reagent used in theTECHNICON IMMUNO 1 system were aspirated into the first segment (MP),with the remainder of fluid being particle wash buffer (25 mM Tris, pH7.5, containing 0.2 M NaCl, 0.1% Triton X-100 and preservative). R1(10.4 μl of serum-based solution containing fluorescein-labeled antibodyto TSH), R2 (10.4 μl of serum-based solution containing antibody to TSHconjugated with ALP) and S (7.2 μl of serum sample) were aspirated intothe second segment. The next two segments (W1 and W2) were comprised ofthe same wash buffer used above in the MP segment. The fifth segment wasR3, of the composition described above, with the key elements being theluminogenic substrate and the luminescence enhancer. The sixth segmentwas an inter-test buffer (same as the particle buffer described above),which was used to isolate adjacent tests. Magnetic transfers aredepicted by the arrows in the FIG. 1. These transfers were facilitatedby one of two magnetic transfer assemblies (M1 or M2). After anincubation of 13 minutes, during which sandwich formation occurred, M1transferred the magnetic particles into the R1+R2+S segment to initiatecapture. After an additional period of 6 minutes, M2 transferred theparticles into the first wash segment. After an additional period of 12seconds, M2 transferred the particles into the second wash segment.After another period of 12 seconds, M2 transferred the particles intothe R3 segment, and light emission from this segment was detected as thestream of aqueous segments passed back and forth through the luminometerreadhead.

Since the Teflon tube is transparent to light, a problem with lightpiping (or “optical carryover”) was expected. Specifically, some of thephotons emitted from the R3 segment of an adjacent test could enter theTeflon material, propagate down the length of the tube and be scatteredinto the detector during the measurement of the signal of the test ofinterest. However, while a signal was detected in the adjacent tests, itdid not occur in the expected manner. Instead of declining rapidly withdistance from test N, peaks of light output were observed centeredaround the R3 segments of the adjacent test packages, as shown in FIG. 2of the drawings. In FIG. 2, test N produced a high level ofluminescence, approximately 7.5 million counts per seconds (cps). TestsN−1 and N−2 were aspirated into the tube before test N and preceded thistest through the luminometer, and tests N+1 and N+2 followed after testN. The analysis system recorded photons counted for each individual airand liquid segment in the stream. The profile in FIG. 2 represents theaverage of 10 replicate panels of 5 tests each corrected for backgroundluminescence signal produced in the absence of ALP. The reagent blankvalues subtracted from each data point were an average obtained from 10replicate panels of 5 tests each. The magnitude of the carryover signalwas computed by dividing the peak cps in each adjacent test by the peakcps in test N, expressed in parts per million (ppm).

Another possible explanation for this behavior was physical carryover ofALP from test N into the neighboring tests in an unintended manner. Thiscould happen, for example, if the tube contained particulate materialsdeposited on the walls, which could disrupt the smooth motion of theliquid segments through the tube. However, placement of 10 mM inorganicphosphate in the R3 segments of the adjacent tests had no effect on themagnitude of the signals in the adjacent tests. Since this amount ofphosphate would have inhibited ALP by at least 90% under these testconditions, the possibility of physical carryover was ruled out.

To further rule out optical carryover, the fluorescent enhancer pyraninewas omitted from test N only, but present in the adjacent tests. As aresult, the magnitude of the signal in test N was lower by a factor ofapproximately 10. However, as shown in FIG. 3 of the drawings, theheight of the peaks in the adjacent tests did not change significantly.The fact that the carryover signal did not change in the adjacent testsproportionately clearly demonstrated that this carryover was notoptical.

An additional and unexpected type of carryover was the cause of thecarryover problem. It was found that the hydroxy dioxetane intermediatewas sufficiently soluble in the fluorocarbon oil used to coat the innerwall of the Teflon tube, such that the carryover was due to transfer ofdissolved hydroxy dioxetane intermediate via the oil into the R3segments of the neighboring tests. This process was tested by changingthe buffer of the R3 segments in the adjacent tests from 1 M DEA at pH10 to 1 M Tris at pH 7. At pH 7, dissolved hydroxy dioxetaneintermediate in these R3 segments is stable and does not emit light. Asshown in FIG. 4 of the drawings, this change in pH resulted in thecomplete elimination of the side bands of luminescence. The residualminor carryover in the N+1 and N−1 tests was due to the anticipatedoptical carryover. These results verified that the source of lightemission in the peaks in the neighboring tests was “chemical carryover”of the hydroxy dioxetane derived from CSPD into the R3 segments ofadjacent tests.

Example 8 Elimination of Observed Chemical Carrvover with DicarboxylicAcid-Substituted Dioxetane 1

Table 1 shows the effect of using three other dioxetanes on the chemicalcarryover of the reaction intermediate. LUMIGEN PPD[4-(methoxy)-4-(3-phosphoryloxyphenyl)]spiro-[1,2-dioxetane-3,2′-tricyclo[3.3.1.1^(3,7)]-decane],(Lumigen, Inc., Southfield, Mich., USA), dioxetane 2, a monocarboxylicacid derivative and dioxetane 1, a dicarboxylic acid derivative wereeach used in test formulations at the same concentration. The ppm columnis the signal for the N+1 test, which represents worst case behavior.The carryover of the unmodified parent compound, PPD, was found to bemore than twice as high as that observed with CSPD. Surprisingly, themonocarboxylic acid derivative, dioxetane 3, showed a reduction of only84% in the magnitude of the chemical carryover. This indicated that asingle charged group was insufficient to completely preventsolubilization of the reaction intermediate in the fluorocarbon oil.However, the dicarboxylic acid derivative was 100% effective, indicatingthat two charged groups were fully adequate to achieve the desiredbehavior.

TABLE 1 Reduction of Chemical Carryover Compound ppm % Reduction LUMIGENPPD 1640 Dioxetane 2 260 84 Dioxetane 1 0 100

Example 9 The Role of Enhancers

As part of the optimization of a reagent based on dioxetane 1, a numberof enhancer materials was examined. At pH 9.6, Enhancer A(1-trioctylphosphoniummethyl-4-tributylphosphoniummethylbenzenedichloride) increased the luminescent signal by a factor of 6.2, andEnhancer B (poly(vinylbenzyltributylphosphonium chloride)) increased thesignal by a factor of 19.7. At pH 10.0, Enhancer A increased the signalby a factor of 4.8, and Enhancer B increased the signal by a factor of18.9.

Despite the fact that Enhancer B achieved higher light intensities,Enhancer A was preferred for use on the analysis system since it is alow molecular weight monomeric compound. Polymeric compounds, especiallyif they are polycationic, interact with serum components, causingprecipitation, which would pose significant problems for the operationof the analysis system.

Both fluorescein and pyranine were found to be effective assupplementary fluorescers in combination with Enhancer A. Alone, thesefluorescers must be used at relatively high concentrations (3 mM) inorder to achieve an enhancement of about ten-fold. However, incombination with Enhancer A, a synergistic effect was observed, in whicha comparable enhancement resulted at 100-fold lower concentrations offluorescer than needed in the absence of the enhancer. Tables 2 and 3show the extent of enhancement by pyranine and fluorescein,respectively, in the presence of 1 mg/mL of Enhancer A.

TABLE 2 Enhancement by Pyranine with Enhancer A [Pyranine] (mM)Enhancement Factor 0.01 3.7 0.02 7.3 0.03 9.8 0.04 12.2 0.05 13.7

TABLE 3 Enhancement by Fluorescein with Enhancer A [Fluorescein] (mM)Enhancement Factor 0.01 2.6 0.02 4.0 0.05 7.1 0.10 8.7

Example 10 Optimized Formulation for Capsule Chemistry Analysis System

The above described observations have led to the development of anoptimized formulation for the capsule chemistry analysis system. Thisformulation is comprised of 0.1-1 mM dioxetane 1, 0-0.05 mM pyranine,0.1-5 mg/mL Enhancer A, 0-1 mM Mg⁺², 0.1-1 M 2-amino-2-methyl-1-propanol(pH 10.0) and 0.01-1% Triton X-100. Use of this formulation results incomplete elimination of the chemical carryover problem and enhancedperformance.

Example 11 Stability of 1, 3, 4 and 5 Measured by Enzyme Assay

Formulations comprising 0.1 mg/mL Enhancer A, 0.88 mM Mg⁺² , 0.2 M2-amino-2-methyl-1-propanol, pH 10, 0.1% Triton X-100 and 0.5 mMdioxetane 1, 3, 4 and 5, respectively, were prepared and stored inopaque polyethylene bottles at 4° C., 25° C. and 40° C. Twenty four 100μL aliquots from each bottle were pipetted into the wells of a 96 wellplate and the solutions incubated at 37° C. Into each well 10 μLsolutions containing 8×10⁻¹⁷ moles of AP were injected and lightintensity integrated over five hours. Data are the average of all 24wells. The experiment was repeated at the indicated time intervals foreach dioxetane. The results in FIG. 5 show the comparative stability ofthe three formulations at 25° C. As shown in FIG. 5, fluoro-substituteddioxetane 3 was found to exhibit substantially better storage stabilitythan chloro-substituted dioxetane 4 and non-halo-substituteddioxetane 1. Dioxetanes 3 and 5 were also substantially more stable than1 or 4 at 40° C.

TABLE 4 Storage Stability of Formulations % of Dioxetane Remaining Time(wks) 1 3 4 5 0 100 100 100 100 1 94.8 100 2 91.1 77.0 99.8 3 87.5 99.166.0 4 84.1 65.6 99.4 5 81.8 6 80.7 9 76.5 96.9 10 57.5 12 96.7 14 93.821 93.6

Example 12 Performance of 3

A detection reagent incorporating dioxetane 3 was evaluated in a testsystem as described in Example 7. The test material was afluorescein-labeled alkaline phosphatase conjugate which was capturedonto the magnetic particles. Assays for AP using the reagent containing3 produced results with sensitivity, dynamic range and precisioncomparable to the results using dioxetane 1.

The foregoing examples are illustrative only and not intended to berestrictive. The scope of the invention is indicated only by theappended Claims and equivalents.

What is claimed is:
 1. A dioxetane of the formula:

wherein R₃ and R₄ are each selected from the group consisting ofacyclic, cyclic and polycyclic organic groups which can optionally besubstituted with heteroatoms and which can optionally be joined togetherto form a cyclic or polycyclic ring group spiro-fused to the dioxetanering, wherein R₂ is an aryl ring group selected from the groupconsisting of phenyl and naphthyl groups which can include additionalsubstituents, wherein Z is selected from the group consisting of afluorine atom and an alkyl group of 1-4 carbons, wherein each R′ is analkyl group of 1-4 carbons and each Y is selected from halogen atoms,substituted or unsubstituted alkoxy, aryloxy, aralkyloxy andtrialkylsilyloxy groups.
 2. The compound of claim 1 wherein Z is F andeach Y is a substituted alkoxy group.
 3. The compound of claim 1 whereineach Y is a 2-cyanoethoxy group.
 4. The compound of claim 1 wherein Z isCH₃ and each Y is a substituted alkoxy group.
 5. The compound of claim 4wherein each Y is a 2-cyanoethoxy group.
 6. The compound of claim 1wherein Z is F and each Y is a chlorine atom.
 7. The compound of claim 1wherein Z is CH₃ and each Y is a chlorine atom.
 8. A dioxetane of theformula:

wherein R₃ and R₄ are each selected from the group consisting ofacyclic, cyclic and polycyclic organic groups which can optionally besubstituted with heteroatoms and which can optionally be joined togetherto form a cyclic or polycyclic ring group spiro-fused to the dioxetanering, wherein R₂ is an aryl ring group selected from the groupconsisting of phenyl and naphthyl groups which can include additionalsubstituents, wherein Z is selected from the group consisting of afluorine atom and an alkyl group of 1-4 carbons, and each R′ is an alkylgroup of 1-4 carbons.
 9. The compound of claim 8 wherein Z is F.
 10. Thecompound of claim 8 wherein Z is CH₃.